Voltage-gated calcium channels (CaV) are key regulators of cellular excitability; they translate electrical information into biochemical responses in excitable cells such as nerve and muscle cells. CaV are separated in three families: CaV1, CaV2 and CaV3. CaV1 and CaV2 typically comprise a pore-forming alpha1 with auxiliary β and alpha2delta subunits. The alpha2delta enhances surface expression and modulates the biophysical properties of CaV. It has been implicated in pain and epilepsy, and the target for anti-epileptic and anti-nociceptive gabapentinoid drugs. Despite its clinical significance, the relationship between the structure and function of this subunit remains poorly understood. Fitzgerald and co-workers recently showed that the N-terminal region of alpha2delta-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length alpha2delta on CaV2.2 channels. In order to understand the functional role(s) of Rd and the regions downstream of it, the biochemical and cell biological properties of alpha2delta were explored producing a set of alpha2delta-truncated proteins, in which the delta protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the alpha2delta-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the delta protein, were prepared after successive additions of stop codons (TGA) in the alpha2delta (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of alpha2delta-1/-2 has been suggested to be critical for trafficking of alpha2delta to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of alpha2delta, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of alpha2delta at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-alpha2) on CaV2.2/β1b channels revealed that, in contrast to the full functionality of Rd alone, extension to the end of the VWA domain, or the alpha2 region, abolished typical alpha2delta-mediated current enhancement. Nevertheless, both constructs increased rate of voltage-dependent inactivation, indicating that they interact with the channel via Rd. Thus, Rd appears to contain all the machinery required to support the electrophysiological and trafficking effects of alpha2delta. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 alpha1 subunit that interact with the Rd. Such an approach could be applied to other alpha1 subtypes to determine discrete alpha2-Rd interactions, information that is critical for further therapeutic exploitation of alpha2delta. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how alpha2delta interacts with CaV.

Les récepteurs du glutamate de type AMPA (rAMPA) sont les récepteurs ionotroniques responsables de la majeure partie des courants excitateurs rapides lors de la transmission synaptique dans le système nerveux central. Le nombre de rAMPA stabilisés à la synapse est responsable en partie de l’intensité de la transmission synaptique et de nombreux phénomènes de plasticité synaptique. Les rAMPA se répartissent en trois populations en équilibre dynamique: les récepteurs intracellulaires, les récepteurs extra-synaptiques, et les récepteurs synaptiques stabilisés au niveau de la densité post-synaptique. L’implication des protéines transmembranaires régulatrices des rAMPA (TARP) dans la stabilisation des rAMPA est établie, et repose au moins en partie sur la liaison de la protéine TARP γ-2 avec la protéine d’échafaudage PSD-95. Dans l’hippocampe, siège de nombreux phénomènes de plasticité, l’isoforme γ-8 est particulièrement enrichie. La TARP γ-8 a pour particularité de posséder un domaine C-terminal plus long que son homologue γ-2 et de s’exprimer au niveau synaptique et extra-synaptique. Mon travail de thèse à consisté à étudier les mécanismes moléculaires mis en jeu dans la régulation de la liaison des protéines TARP γ-2 et γ-8 avec la protéine PSD-95, ainsi que l’implication respective des deux isoformes dans la régulation de la mobilité latérale des rAMPA. Les résultats majeurs de cette étude sont : a) l’interaction entre γ-2 et PSD-95 est régulée par la longueur apparent du domaine C-terminal de γ-2 modulée par la phosphorylation; b) γ-8 lie PSD-95 dans les compartiments synaptiques et extra-synaptiques, toutefois cette interaction n’est pas corrélée avec une immobilisation des rAMPA. Ces résultats suggèrent que γ-2 et γ-8 jouent des rôles bien distincts dans l’adressage des rAMPA à la synapse. / AMPA type glutamate receptors (AMPARs) are ionotropic receptors responsible for most excitatory transmission in the central nervous system. The number of stabilized AMPARs in front of glutamate release sites determines in large part the strength of synaptic transmission and variation in this number is thought to underlie numerous forms of synaptic plasticity. AMPARs are present in three main subcellular pools between which they are in a dynamic equilibrium by processes of trafficking: intracellular receptors, extrasynaptic receptors, and synaptic receptors stabilized at the postsynaptic density (PSD). Transmembrane AMPAR regulatory proteins (TARPs) are known to be implicated in AMPAR stabilization at the synapse through the interaction of TARP γ-2/8 with the scaffolding protein PSD-95. In the hippocampus, a structure exhibiting various synaptic plasticity patterns, γ-8 is the most abundant TARP. This isoform is characterized by a longer C-terminal fragment than γ-2 and a synaptic and extrasynaptic localization. During my Ph.D, I studied the molecular mechanisms involved in the regulation of TARP γ-2 and γ-8 binding to PSD-95 and their respective roles in regulating AMPAR lateral mobility. The main results are: a) γ-2 interaction with PSD-95 is regulated by the apparent length of its C-terminus domain that is modulated by phosphorylation; b) γ-8 binds PSD-95 in synaptic and extrasynaptic compartment however this interaction is not correlated with AMPAR immobilization. Altogether, those results suggest that those two TARP isoforms have independent functional roles.